Summary of Work: The long-term goals are to identify and characterize key mechanisms that control development or function of germ cells in the male. The approaches being used are to identify genes expressed specifically in male germ cells, to use transgenic mice and in vitro assays to identify promoter elements and transcription factors regulating their expression, and to apply the gene knockout approach to define the roles of the proteins they encode. Many genes are expressed only in male germ cells and we are focusing on a few likely to be important in gamete development or function. We isolated the mouse and human genes for a key isozyme in the glycolytic pathway, glyceraldehyde 3-phosphate dehydrogenase (GAPD-S), that is expressed only in male germ cells and believed to have a key role in regulating generation of ATP required for fertilization. Antibodies prepared to synthetic peptides localized GAPD-S to the fibrous sheath of the sperm flagellum in mouse and human. Yeast two-hybrid screens identified a WW-domain protein (FBP11) which binds to a proline-rich region of mouse GAPD-S. We are mapping the binding domains on each protein and testing the hypothesis that FBP11 anchors GAPD-S to the fibrous sheath. In addition, molecular modeling studies indicate that residues surrounding the substrate-binding pocket may account for differences in the effects of the inhibitor on the somatic and germ cell isozymes (collaborator Bienstock). Human recombinant GAPD-S is being expressed for use in NMR studies to examine the interaction of GAPD-S with its natural substrate, natural cofactor, and reproductive toxicants (collaborator London). One of these is (S)-3-chlorolactaldehyde, a metabolite of the industrial solvent epichlorohydrin that appears to act as a competitive inhibitor of substrate binding to GAPD-S. Furthermore, a Gapds gene-targeting construct is being prepared to test the hypothesis that sperm from knockout mice will be unable to produce the ATP necessary to achieve hyperactivated motility and will be unable to fertilize eggs (collaborators Bunch and O?Brien).We demonstrated previously that fibrous sheath component 1 (FSC1) was the major cytoskeletal protein in the sperm flagellum and that GAPD-S and FSC1 co-localize in this region of the mouse sperm. To study their possible interactions and the role of FSC1, the Fsc1 cDNA and gene were isolated and yeast two-hybrid assays were used to identify associated proteins. FSC1 was found to be a protein kinase A (PKA) anchoring protein (AKAP), suggesting that PKA anchored to the fibrous sheath participates in protein prosphorylation events essential for the activation of sperm motility. Yeast two-hybrid assays, alanine and valine scanning-mutagenesis, and pull-down assays were used to define the amino acids responsible for specificity of binding of different regulatory subunits of the PKA tetramer. RI&#61474;-specific and dual RI&#61474;/RII&#61474;-specific binding motifs identified in FSC1 were the first to be characterization in an AKAP. It was found that hydrophobic amino acids at three consensus positions on AKAPs are required for PKA binding and that specificity of PKA binding is determined by the size of the aliphatic side-chain on the amino acid in the middle position. This was verified by introducing point mutations into these motifs to switch between RI&#61474;-specific, RII&#61474;-specific, and RI&#61474;/RII&#61474; dual-specific binding. These findings represent an important advance in understanding the relationship between the primary sequence and the three-dimensional spatial distribution of residues within the amphipathic &#61474;-helix of AKAP anchoring domains that determine PKA binding specificity. They are also significant for advancing the understanding of molecular mechanisms involved in PKA subtype localization within cells.Other studies are using yeast two-hybrid screens to identify additional proteins that bind to GAPD-S and to FSC1 to determine how these and other proteins we identified previously assemble into a fibrous sheath. The proteins include male germ cell-specific isoforms of hexokinase 1 (HK1-S), glutathione S-transferase (GSTM5), AKAP110, and two unknown proteins. Gene targeting is also being used to produce Fsc1 gene knockout mice that are predicted to be infertile due to disruption of flagellar structure and function. They will be crossed with transgenic mice that express FSC1 with point mutations that abolish RI&#61474; and RII&#61474; binding or that result in switches in PKA isoform binding. The transgenes will be under the control of a promoter expressed only in spermatogenic cells. These animal models will allow in vivo studies of the function of FSC1 and of the role of different PKA isoforms in sperm function, as well as the analysis of the interactions between AKAPs and PKAs.We used gene targeting to disrupt expression of fertilin &#61538;, a sperm surface molecule that is the archetype of the ADAM (a disintegrin and metalloprotease domain) family of proteins involved in cell-cell interactions (collaborators Myles and Primakoff). The fertilin &#61537;:&#61538; dimer participates in sperm-egg interaction and binds to a &#61537;6&#61538;1 integrin on the egg surface. Male fertilin &#61538; knockout mice produce typical numbers of motile sperm and show normal mating behavior, but are infertile because few sperm enter the oviduct and those that do are unable to bind to eggs. It appears that loss of metalloprotease activity alters sperm-oviduct interaction and that loss of disintegrin activity disrupts sperm-egg binding. We also are using gene targeting to disrupt expression of protamines 1 and 2, two other important sperm proteins (collaborator Hecht). The protamines are highly basic nuclear proteins that replace the histones following meiosis and are thought to be essential for DNA compaction in spermatids, a cell type that is haploid and lacks nucleosomes. It is hypothesized that disruption of one or both protamine genes will lead to abnormal nuclear compaction and infertility in homozygous males.